GSM6619548: HCT17HEX, scRNAseq; Homo sapiens; RNA-Seq
Approximately 50-100 mg of frozen tissue per sample was homogenized in 4 mL of nuclei extraction buffer [0.32 M sucrose, 10 mM Tris pH 7.4, 5 mM CaCl2, 3 mM Mg acetate, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton X-100, 0.2U/uL Protector RNAse inhibitor (Sigma cat. 3335399001)] by douncing 30 times in a 40 mL dounce homogenizer. Filter through 70 um filter and spin at 500xg, 5 min at 4°C in a swinging bucket centrifuge. Resuspend nuclei in 500 uL nuclei extraction buffer and layer over 750 uL sucrose solution (1.8 M sucrose, 10 mM Tris pH 7.4, 3 mM Mg acetate, 1 mM DTT) in a 1.5 mL tube. The samples were then centrifuged at >16,000 x g for 30 min at 4°C. After centrifugation, the supernatant was removed by aspiration and the nuclear pellet was resuspended in 125 uL PBS with 1% BSA and centrifuged 5 min at 500 x g at 4°C in a swinging bucket centrifuge. Permeabilization was performed according to 10X Genomics protocol CG000375 Rev B:  nuclei were resuspended in 100 uL lysis buffer (10 mM Tris-HCl pH 7.4,10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.01% Tween-20, 0.01% NP-40, 0.001% digitonin, 1 mM DTT, 1 U/uL Protector RNase inhibitor) and incubated 2 min on ice. Nuclei were washed once and resuspended in 30 uL of 1X nuclei buffer with 1 mM DTT and 0.5 U/uL of Protector RNAse inhibitor. Nuclei quality and concentrations were determined using the Countess.  Library was performed according to the manufacter's instructions (single cell multiome protocol CG000338 Rev E, 10x Genomics).